Biological membranes regulate fluxes of water and other small molecules over a 3 order of magnitude permeability range, from apical membranes of barrier epithelia (very low permeabilities) to membranes which contain water channels (very high permeabilities). The original grant and this renewal proposal seek to answer two physiological questions: 1. What are the structural features of barrier epithelial apical membranes which restrict the fluxes of water, urea and ammmonia? 2. How does the pore of aquaporins (AQPs) function, and how are the primary structures of the different AQPs related to pore function? Aim #1 will determine how the structure of barrier epithelial apical membranes restricts permeability. Studies using asymmetrie artificial bilayers in which the composition of each - leaflet differs will be used to test the conclusion that each leaflet offers an independent resistance to permeation. The apical membrane structure of MDCK cells, a model barrier epithelium, will be determined. Using this information the lipid structure of artificial bilayers and the MDCK cell apical membrane will be sysmmatically modified and the effects of these modifications on permeabilities determined. These studies will define the role of individual lipid substituents in apical membrane permeability. Aim #2 will examine the relationship between AQP primary sequence and the pore properties of AQPs including water conductance, pore configuration, selectivity for water over other substances, and regulation by phosphorylation. The pore properties of AQP 1, AQP 2, AQP 3, AQP 0, and NOD 26 (a model AQP from plant root nodules) will be defined because these AQPs appear to represent the range of AQP pore properties currently known. These studies will be performed in native membranes (such as red blood cells and water channel containing vesicles from collecting duct), in proteoliposomes with reconstituted AQPs and in a newly developed heterologous expression system, sec6 vesicles. These studies will define the nature of the AQP pore and will begin to relate AQP primary structure to pore function, setting the stage for detailed mutagenesis studies in the future. Building on the substantial progress achieved in the first funding period, this competing renewal proposal seeks to define, at a molecular level, the mechanisms by which water and other small molecules cross low permeability lipid bilayers and water channels.